0

1st Satellite Seminar

of

New Core to Core Program

Establishment of an international research core for new

bio-research fields with microbes from tropical areas

(World-class research hub of tropical microbial resources and

their utilization)

Organized By

University of Brawijaya (UB)

In association with

Japan Society for the Promotion of Science (JSPS)

National Research Council of Thailand (NRCT)

Vietnam Ministry of Science and Technology (MOST)

Yamaguchi University, Kasetsart University, Can Tho University, National

University of Laos, Beuth University of Applied Science, The University of

Manchester

8th

August 2014

5th

Floor of East Java Bank, Surabaya

1

Abstract Book of The 1st Satellite Seminar of CCP, 8 August 2014,

Venue: 5th

Floor, East Java Bank, Surabaya-Indonesia

Page Note

Cover of Abstract book

I Message from Indonesia Coordinator

II Message from Japan Coordinator

III Message from Thai Coordinator

V Message from Vietnam Coordinator

VI Message from UK Coordinator

VII Committee

VIII Seminar Program

2

Message from Indonesia Coordinator Dr. Anton Muhibuddin

Dear All,

Firstly, I introduce Indonesia as a new member of Core to Core

Program (CCP). Thank you very much for Prof. Mamoru Yamada, as a

coordinator of CCP for Japan side who has facilitated the participation

of Indonesian scientist in this program. I hope, participation of

Indonesian scientist can support CCP programs better. As a new

member of CCP, it is not easy to run this program as well as Thailand

and Japan which has run since 1987. This is due to the schedule of

academic and research funding are different in Indonesia. An example is

that the budget submission for the program in Indonesia is limited by

time up to the end of March every year. However, of course Indonesia

will try best to support this program. As the country with the third

largest population in the world, the number of scientists in Indonesia is

also very big and potential to support development of science in the

world. Moreover, the quality and quantity of our natural resources is

very great to support the development of world’s science.

Overall, I am delighted to welcome all of the speakers and participants to the 1st Satellite

Seminar of the New Core to Core Program A. Advanced Research Networks on “Establishment of

an International Research Core for Bio-research Fields with Microbes from Tropical Areas (World-class

Research Hub of Tropical Microbial Resources and Their Utilization)”.

On behalf of Indonesia Coordinator, I would like to express my sincere appreciation to all

participants for contributing their research work to this seminar. Thanks also to the Japanese,

Thailand, Vietnamese, Laotian, Germany and UK coordinators for their cooperation in arranging

this seminar. I also would like to express sincere gratitude to East Java Bank, University of

Brawijaya, DIKTI, University of KH. Abd. Wahab Hasbullah, JSPS, NRCT, Vietnam Ministry of

Science & Technology (MOST), the National University of Laos, Beuth University of Applied Sciences

(Germany) and The University of Manchester (England) for their financial support.

Kind regards,

Anton Muhibuddin Indonesia coordinator

3

Message from Japanese Coordinator Prof. Mamoru Yamada

It is our great pleasure to hold the 1st Satellite Seminar in the

Core-to-Core Program (Advanced Research Networks) entitled

“Establishment of an international research core for new bio-research

fields with microbes from tropical areas (World-class research hub of

tropical microbial resources and their utilization)”. I would like to take

this opportunity to acknowledge the enormous effort of the organizing

committee members of this seminar at the Indonesia side, especially Dr.

Anton Muhibuddin and his colleagues, and the financial support of University of Brawijaya.

We have launched the five-year Core-to-Core Program, in which many scientists participate

from seven countries of Thai, Vietnam, Laos, Germany, Indonesia, United Kingdom and Japan. For

the approval of this program, we would like to appreciate tremendous activities and highly valuable

achievements of the members in the ten-year JSPS-NRCT Core University Program entitled

“Development of Thermotolerant Microbial Resources and Their Application” and in the five-year

JSPS-NRCT Asian Core Program entitled “Capacity Building and Development of Microbial

Potential and Fermentation Technology towards New Era”. The Core-to-Core Program is designed

to create top world-class research centers that partner over the long term with other core research

institutions around the world in advancing research in leading-edge fields, on issues of high

international priority. Thus, we should challenge the new bio-research fields with microbes from

tropical areas.

In our Core-to-Core Program, there are the following five projects, which will be performed

by internationally collaborative researches among seven countries. I thus hope members at

Indonesia side to find out good counterparts to work together on original research topics related to

these projects. During this seminar, you may obtain beneficial information or new ideas from

presentation and discussion, which promote your further experiments.

Project 1: Explorational Research of Useful Microbes

Project 2: Genome-based Research on Thermotolerant Microbes

Project 3: Research on Environmental Microbes Sustaining Tropical Ecosystem

Project 4: Research on Microbes Useful for Food, Packaging, Health, and Ecosystem

Project 5: Development of Next-generation Fermentation Technology for New Wave

After this seminar, the 1st Joint Seminar in Thailand, which is organized by Associate Professor

Dr. Gunjana Theeragool and members at the Thai side, and the 10th Young Scientist Seminar in

Japan, which is organized by young scientists, are scheduled on Aug 10-11 and Nov 16-17 this

year, respectively. This new Core Program is thus expected to contribute not only to microbial

sciences but also to education to foster our successors.

Finally, I would like to appreciate all attendants and their contributions to this seminar, and

the financial supports of University of Brawijaya, National Research Council of Thailand (NRCT)

and Japan Society for the Promotion of Science (JSPS).

Mamoru Yamada Japanese Coordinator Professor, Yamaguchi University

4

Message from Thai Coordinator

Assoc. Prof. Dr. Gunjana Theeragool

I am delighted to welcome all of the distinguished guests and participants to the 1

st Satellite

Seminar of the New Core to Core Program A. Advanced Research Networks on “Establishment of

an International Research Core for Bio-research Fields with Microbes from Tropical Areas (World-class

Research Hub of Tropical Microbial Resources and Their Utilization)”.

Kasetsart University and Yamaguchi University established the Core University Program with

financial support from the Japan Society for the Promotion of Science (JSPS). It took place over 10

years (1998-2007). The success of the 10 year core university program had the potential to be extended

to the Asian Core Program. This program was created with financial support from JSPS and the National

Research Council of Thailand (NRCT), ran for 5 years (2008-2012) and received collaboration from 4

active teams from Japan, Vietnam, Laos and Thailand respectively. Following on from this fruitful

collaboration, we have established the Core to Core Program A. Advanced Research Networks. This 5

year (2014-2018) program receives financial support from JSPS, NRCT, the Vietnam Ministry of

Science & Technology (MOST), the National University of Laos, The University of Brawijaya, Beuth

University of Applied Sciences (Germany) and The University of Manchester (England).

This 1st Satellite Seminar of the Core to Core Program is the second academic activity

arranged after the successful JST workshop on Advanced Low Carbon Biotechnology which was

held at Kasetsart University during the period July 3-4, 2014. This seminar will provide a good

opportunity for all of the participants to meet and discuss their future areas of collaboration in order

to obtain the most fruitful results. In addition, I hope that the presentations and discussions which

take place during this seminar will spur the participants towards the development of new research

opportunities and productive collaboration. With our hard work and contributions, I am sure that

this Core to Core Program will be another successful project, similar to our previous Core

University Program and Asian Core Program.

On behalf of Thai Coordinator, I would like to express my sincere appreciation to The

University of Brawijaya especially Dr. Ir. Anton Muhibuddin, Indonesian Coordinator, for

organizing the 1st Satellite Seminar. My thanks also go out to the invited speakers and all of the oral

and poster presenters for contributing their research work to this seminar. Thanks also to the

Japanese, Vietnamese, Laotian, Indonesian, German and English coordinators for their cooperation

in arranging this 1st Satellite Seminar. Last, but not least, I would like to express sincere gratitude to

JSPS and NRCT for their continuing financial support.

Gunjana Theeragool Thai coordinator and Chairperson of the Organizing Committee

5

Message from Vietnamese Coordinator

Assoc. Prof. Dr. Ngo Thi Phuong Dung

With all pleasure, I am very delighted to welcome all of the distinguished participants to the

1st Satellite Seminar of the Core to Core Program on “Establishment of an international research

core for new bio-research fields with microbes from tropical areas”, hosted by University of

Brawijaya, held on 8th August 2014 at University of Brawijaya, Malang-Indonesia.

The year 2014 has officially marked the great success to start a new Core to Core Program

(April 2014- March 2019) as our wish to further develop our collaboration on science and education

after the Asian Core Program (April 2008- March 2013) on “Capacity building and development of

microbial potential and fermentation technology towards New Era”, which was financially

supported by Japan Society for the Promotion of Science and National Research Council of

Thailand.

It is a great honor and pleasure for the Vietnamese team to be available to continue our

participation in this new program of Advanced Research Networks on “Establishment of an

international research core for new bio-research fields with microbes from tropical areas” – World-

class research hub of tropical microbial resources and their utilization. Our team is also very happy

to have a good opportunity to join with more counterparts from Japan, Thailand, Laos, Germany,

Indonesia, United Kingdom and Vietnam.

May I take this occasion to express a sincere thanks to the support institutions of all partner

countries, and I would like to acknowledge the excellent effort of the organizing committee and

team, especially University of Brawijaya. We are also grateful to all keynote lecturers, oral speakers

and poster presenters as well as all participants who significantly contribute to the success of the

seminar event.

Ngo Thi Phuong Dung Vietnamese Coordinator

Associate Professor, Deputy Director

Biotechnology R & D Institute, Can Tho University

6

Message from UK Coordinator

Prof. Dr. Colin Webb

It has been, for me, a great pleasure to join the Core to Core Programme led by Prof. Dr. Mamoru

Yamada of Yamaguchi University in Japan and to participate in the 1st Joint Seminar on “Capacity

Building and Development of Microbial Potential and Fermentation Technology towards New Era”

being held in Bangkok, Thailand.

Unfortunately, I am unable to participate in the 1st Satellite Seminar of 2014, organized by the

University of Brawijaya (UB) to be held in Malang-Indonesia on 8th August. This seminar promises

to present a wide ranging programme discussing all topics of the core-to-core project. I would like

to pass on my sincerest best wishes for a successful and productive day. I hope also that I, or

members of my team, will be able to participate in future Satellite Seminars.

Colin Webb

UK Coordinator

Professor, University of Manchester

7

Schedule and Program of The 1st Satellite Seminar

New Core-to-Core Program

8th

August 2014 at 5th

Floor of East Java Bank Surabaya – Indonesia

Time Detail Speaker

August 6, 2014

09:40 – 13:05

16:30 - 17:50

Thai members leave for Suvarnabhumi Airport to Singapore Airport by

SQ973 then connect to Juanda Airport (Surabaya) by MI226

Check-in at IBIS Hotel in Surabaya. Location: Jalan Basuki Rahmat,

Surabaya, Jawa Timur 60271, (031) 5358885

August 7, 2014 (23:00) – 00:35 Japanese Members arrive at Sukarno Hatta International Airport (Jakarta),

then depart to Juanda Airport (Surabaya) by GA332

01:30 - 02:00 Depart for IBIS Hotel in Surabaya and check in. Location: Jalan Basuki

Rahmat, Surabaya, Jawa Timur 60271, phone: (031) 5358885

Late Breakfast

12:00 – 13:00 Lunch

13:00 -15:00 To meet President of Brawijaya University (in Malang or Surabaya)

August 8, 2014

08:00 – 08:30 Depart for 5th

Floor of East Java Bank Surabaya for Satellite Seminar

08:30 – 09:00 Registration

09:00 – 09:30 Opening ceremony

09:30 – 10:00 Introduction of Brawijaya University

Indonesia

Dr. Anton Muhibuddin

10:00 – 10:30 Group Photo and coffee break

10:30 – 11:00 Introduction of Core to Core Program Prof. Dr. Mamoru Yamada

Seminar

Session I

Chairman : Prof. Dr. Poonsuk Prasertsan

Co-Chairman : Prof. Dr. Shinichi Ito

Project I: Explorational Research of Useful Microbes

11:00 – 11:25 Oral I-1: Biomass-degrading enzymes and

value-added products from tropical

strains of Aureobasidium pullulans

Assist. Prof. Dr. Sehanat

Prasongsuk

Chulalongkorn University

11:25 – 12:30 Lunch

12:30 – 12:55 Oral I-2 Isolation and identification of

nitrogen fixing non-simbiotic

bacteria on restoration land with

legume cover crop (lcc) in the area

of Pasirian, Lumajang, East Java

Ir. Tutik Nurhidayati, MS.

Institute Technology of 10th

Nopember Surabaya

8

Time Detail Speaker

Project II: Genome-based Research on Thermotolerat Microbes

12:55 – 13:20 Oral II-1: Analysis of thermotolerant genes

in thermotolerant Zymomonas

mobilis

Assist. Prof. Dr. Tomoyuki

Kosaka

Yamaguchi University

13:20 – 13:45 Oral II-2: Essentiality of respiratory activity

for pentose utilization in

thermotolerant yeast

Kluyveromyces marxianus

Dr. Noppon

Lertwattasanakul

Katsetsart University

Seminar

Session II

Chairman : Prof. Dr. Kenji Matsui

Co-Chairman : Dr. Ir. Marjuki

Project III: Research on Environmental Microbes Sustaining Tropical

Ecosystem

13:45 – 14:10 Oral III-1: Newly recognized food-borne

diseases associated with ingestion

of myxosporean spores in marine

fish

Prof. Dr. Hiroshi Sato

Yamaguchi University

14:10 – 14:35 Oral III-2 The potency of phylloplane saprophytic fungi on shallot as

antagonists against purple blotch

disease(Alternariaporri) in East Java,

Indonesia

Dr. Hery Nirwanto

University of Veteran

Surabaya

14:35 – 14:50 Coffee Break

14:50 - 15:15 Oral III-3 Maximizing the essential roles of

rumen microbes in supplying

nutrients for ruminants

Dr. Ir. Marjuki

University of Brawijaya

Project IV: Research on Microbes Useful for Food, Food Preservation,

Health, and Ecosystem

15:15 – 15:40 Oral IV-1: Development of effective

bioremediation technology

utilizing beneficial biofilms

Prof. Dr. Masaaki

Morikawa

Hokkaido University

Seminar

Session III

Chairman : Dr. Ir. Hery Nirwanto

Co-Chairman : Assist. Prof. Dr. Sehanat Prasongsuk

15:40 – 16:05 Oral IV-2: Microbial diversities in the

chemical and organic agricultural

soils

Prof. Dr. Motoki Kubo

Ritsumeikan University

16:05 – 16:30 Oral IV-3: Exploration of marine bacteria as

an alternative source of enzyme

production for industry and

biodegradation

Assist. Prof. Dr. Jittima

Charoenpanich

Burapha University

16:30 – 16:45 Coffee Break

Project V: Development of Next-generation Fermentation Technology

for New Wave Industry

9

Time Detail Speaker

16:45 – 17:10 Oral V-1: Effects of inoculum size and

reactor type on biohydrogen

production from Palm Oil Mill

effluent under thermophilic

condition

Prof. Dr. Poonsuk

Prasertsan

Prince of Songkhla

University

17:10 – 17:35 Oral V-2: In Vitro thermal adaptation useful

for the development of high-

temperature fermentation

Emeritus Prof. Kazunobu

Matsushita

Yamaguchi University

17:35 – 18:35 Group Discussion Project 1 Leaders: Dr. Shinichi Ito

Dr. Sehanat Prasongsuk

Project 2 Leaders: Dr. Mamoru Yamada

Dr. Noppon Lertwattasanakul

Project 3 Leaders: Dr. Anton Muhibuddin

Dr. Tomoyuki Kosaka

Project 4 Leaders: Dr. Kenji Matsui

Dr. Jittima Charoenpanich

Project 5 Leaders: Dr. Poonsuk Prasertsan

Dr. Naoya Kataoka

18:35 – 19:00 Closing Ceremony

19:00 – 21:00 Dinner

August 9, 2014

08.00 Thai member check out and depart for Juanda

Airport Surabaya

08:30 – 10:30 Check in ticket and immigration office in

Juanda Airport Surabaya

10:30 – 13:40 Depart for Singapore Airport by SQ931

16:00 – 17:25 Depart for Suvarnabhumi Airport Bangkok

by SQ976

10:00 - 10:30 Japanese members check out and depart for

Juanda Airport Surabaya

10:30 – 11:30 Lunch

11:30 – 13:30 Check in ticket and immigration office in

Juanda Airport Surabaya

13:30 – 15:05 Depart for Sukarno Hatta Airport Jakarta by

GA317

16:40 – 20:10 Depart for Suvarnabhumi Airport Bangkok

by GA864

Note: Oral presentation for 20 min and discussion for 5 min

10

Biomass-degrading enzymes and value-added products

from tropical strains of Aureobasidium pullulans

Sehanat Prasongsuk1, Wichanee Bankeeree

1, Benjawan Yanwisetpakdee

1, Rinji Akada

2, Pongtharin

Lotrakul1, and Hunsa Punnapayak

1

1Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science,

Chulalongkorn University, Bangkok 10330, Thailand 2Department of Applied Molecular Bioscience, Division of Engineering, Yamaguchi University Graduate

School of Medicine, Ube 755-8611, Japan

A number of Aureobasidium pullulans strains were obtained from various habitats

in Thailand and were identified using morphological observation and molecular

characterization. These strains were evaluated for their production of biomass-degrading

enzymes, mainly -xylanase and -xylosidase, and value added products including

exopolysaccharide (pullulan and -glucan), antifungal agent and siderophores. Some of

these tropical isolates were able to produce cellulase-free xylanase which as applicable in

pulp bleaching. The produced xylanase was also used for degrading xylan, extracted from

various tropical weeds, in order to produce a functional prebiotic, and xylooligosaccharides.

The full-length gene of -xylosidase, an enzyme responsible for xylobiose degradation

from a tropical strain of A. pullulans was revealed. Moreover, the abilities of these A.

pullulans strains for the production of pullulan, -glucan, antifungal agent and siderophores

were also investigated.

11

ISOLATION AND IDENTIFICATION OF NITROGEN FIXING NON SIMBIOTIC

BACTERIA ON RESTORATION LAND WITH LEGUME COVER CROP (LCC)

IN THE AREA OF PASIRIAN, LUMAJANG, EAST JAVA

Tutik Nurhidayati, Nur Hidayatul Alami, and Amik Agisti

Institute Technology of 10th

Nopember Surabaya, Indonesia

Abstrack Application of LCC using peanut (Arachis hypogaea) on critical agricultural land is able to

improve soil fertility. This system produces a good rizosfer that support the bacteria growth. This

research was conducted to determine the abundance and identify bacteria genus of nitrogen fixing

non symbiotic bacteria in land restoration with LCC in Pasirian, Lumajang, East Java.

Bacterial abundance was calculated using Total Plate Count (TPC) method.

Charateristic was to know are bacterial mophological in cultivated medium and

biochemistry test. Determination the bacterial genus was use Bergey’s manual of

determinative Bacteriology 9th edition.

The research results show population of Nitrogen fixing non-symbiotic bacteria on

agricultural land Pasirian before LCC is 3x102 CFU/g and after LCC is 2x103 CFU/g, and

show three bacterial genus of Nitrogen fixing non-symbiotic bacteria found in land

restoration with LCC Azotobacter Beijerinckia, Derxia. Azotobacter are gram-negative and

coccus bacterium, Beijerinckia are gram-negative bacilli, with positive catalase test, and

Derxia is a gram-negative bacilli with negative catalase test.

Keywords: LCC, nitrogen fixing bacteria, non symbiotic.

12

Analysis of thermotolerant genes in thermotolerant Zymomonas mobilis

Tomoyuki Kosaka1, Tomoko Sakurada

2, Amina Tokiyama

1, Kannikar Charoesuk

1,3, Keisuke Hisano

2,

Masayuki Murata2, Mamoru Yamada

1,2

1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan,

2Applied

Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Japan, 3Rajamangala University

of Technology Isan, Thailand

To develop thermotolerant and highly efficient ethanol-producing microbes, the

elucidation of the thermotolerant mechanism is indispensable. Thermotolerant

microorganisms may intrinsically possess the mechanism to prevent the cell damage by

heat stress at a critical high temperature (CHT). In Escherichia coli, 51 thermotolerant

genes and 26 semi-thermotolerant genes, which are required for growth at CHT, have been

identified (1). These genes can be classified into energy metabolism, amino acid

metabolism, vitamin metabolism, outer membrane stabilization, DNA repair, tRNA

modification, and cell division. In this study, identification of the thermotolerant genes in a

relatively thermotolerant ethanol-producing bacterium, Z. mobilis TISTR 548 was

performed to understand the functional commonality and difference of thermotolerant

genes among different microorganisms.

In TISTR 548, 67 thermosensitive mutants at 39.5 °C were screened from ca. 8,000

transposon-inserted mutants. They were then subjected to TAIL-PCR followed by

nucleotide sequencing to determine the corresponding thermotolerant gene. In addition, the

polar effect of transposon insertion on the expression of down-stream genes was examined

by RT-PCR. As a result, 31 thermotolerant genes were identified in TISTR 548. The

identified thermotolerant genes were classified into 9 groups as energy and general

metabolism, membrane stabilization, DNA repair, tRNA modification, chaperon/protease,

translation and translation control, cell division, transcription regulation and others.

1. Murata M, Fujimoto H, Nishimura K, Charoensuk K, Nagamitsu H, Raina S, Kosaka T, Oshima T, Ogasawara N, Yamada M (2011) Molecular strategy for survival at a critical

high temperature in Escherichia coli. PLoS ONE, 6: e20063.

13

Essentiality of respiratory activity for pentose utilization in

thermotolerant yeast Kluyveromyces marxianus

Noppon Lertwattanasakul

1, Nadchanok Rodrussamee

2, Masayuki Murata

3, Sukanya Nitiyon

3,

Suprayogi3, Savitree Limtong

1, Tomoyuki Kosaka

4 and Mamoru Yamada

3,4

1Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand

2Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand

3Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube 755-8505, Japan

4Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515,

Japan

The development of sustainable and renewable biofuels has attracted growing

interests with concerns on increase oil demands and a cleaner environment worldwide. The

economy of fermentation-based bioprocess including bioethanol production relies

extensively on the performance of fermentative microbes.

Kluyveromyces marxianus has been adopted by industries for a relatively broad

range of applications from biomass production to bioremediation due to advantages of its

traits such as rapid growth, thermotolerance, secretion of the enzyme inulinase and

production of ethanol from various carbon sources including glucose, mannose, galactose,

xylose and arabinose. A particularly attractive application of this yeast is high-temperature

fermentation and bioconversion of hemicellulose. The negative effect by coexisting glucose

in the substrates is critical for utilization of biomass containing mixed sugars. In K.

marxianus, glucose repression effect was found to be weaker than that of Saccharomyces

cerevisiae1), 2)

. As a disadvantage, the respiratory activity in K. marxianus becomes strong

when it was grown on pentose sugar, resulting in low production of ethanol. In order to

identify key factors involved in pentose metabolism, experiment on K. marxianus by a

random kanMX4-insertion mutagenesis was performed3)

. We obtained three mutants of

COX15, ATP25 and CYC3 encoding a cytochrome oxidase assembly factor (singleton), a

transcription factor required for assembly of the Atp9p subunit of mitochondrial ATP

synthase and cytochrome c heme lyase, respectively, as mutants lacking growth capability

on xylose and/or arabinose. The three mutants were thermosensitive and their biomass

formation in glucose medium was reduced, but ethanol yields were increased compared to

those of the parental strain. Experiments on the mutants with respiratory inhibitors and

uncoupling agent revealed that the respiratory activity and ATP are essential for utilization

of pentoses. Taken together, this knowledge will be useful to further improve the strain for

high-temperature ethanol fermentation.

References: 1) Rodrussamee N., Lertwattanasakul, N., Hirata, K., Suprayogi, Limtong, S., Kosaka, T. & M.

Yamada. Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect

under various conditions in thermotolerant yeast Kluyveromyces marxianus. Appl. Microbiol.

Biotechnol. 90, 1573–1586 (2011).

2) Lertwattanasakul N., Rodrussamee, N., Suprayogi, Limtong, S., Thanonkeo, P., Kosaka, T. & M. Yamada. Utilization capability of sucrose, raffinose and inulin and its less-sensitiveness to glucose

repression in thermotolerant yeast Kluyveromyces marxianus. AMB Express. 1, 20 (2011).

3) Lertwattanasakul N., Suprayogi, Murata, M., Rodrussamee, N., Limtong, S., Kosaka, T. & Yamada, M. Essentiality of respiratory activity for pentose utilization in thermotolerant yeast Kluyveromyces

marxianus DMKU 3-1042. Antonie van Leeuwenhoek. 103, 933–945 (2013).

14

Newly recognized food-borne diseases associated with ingestion of

myxosporean spores in marine fish Hiroshi Sato

1, Tetsuya Yanagida

1, Takahiro Ohnishi

2, Yoichi Kamata

2,3 and Yoshiko Sugita-Konishi

2,4

1Laboratory of Parasitology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Japan,

2Division

of Microbiology, National Institute of Health Sciences, Japan, 3(Present address) Laboratory of Veterinary

Public Health, Faculty of Agriculture, Iwate University, Japan, and 4(Present address) Laboratory of Food

Hygiene, Department of Food and Life Sciences, Graduate School of Life and Environmental Sciences, Azabu

University, Japan.

Food borne diseases due to unknown causes, manifested as diarrhea and vomiting after

an incubation time from 3.4 to 16.3 hours after consumption of raw marine fish slices

(sashimi), have been noticed since 1999 in the western part of Japan. In 2011, a novel

multivalvulid species parasitic to olive flounder, Kudoa septempunctata Matsukane et al.,

2010 (Myxozoa: Myxosporea), was identified as one of the causative agents of “the

unidentified food borne disease associated with consumption of raw fresh fish” by a

research group supported by the Ministry of Health, Labour and Welfare of Japan (Kawai

et al., 2012). Kudoa septempunctata is morphologically characterized by spores with 6—7

shell valves and polar capsules, localized in the myofiber of trunk muscles of olive flounder

by forming pseudocysts (Matsukane et al., 2010). Based on an epidemiological analysis,

the threshold for the onset of symptom is estimated to be approximately 7.2 x 107 K.

septempunctata spores/person. Pathogenetic mechanisms of K. septempunctata spores were

explored using rodent models (suckling mice and house musk shrews) and a culture model

using human intestinal cell (Caco-2) monolayer.

In additon to K. septempunctata in olive flounder, multiple new myxosporean species in

edible marine fish around Japan have been described by us to list up all possible causative

agents of “Kudoa food borne disease” emerging recently along with the development of

marine fish culture.

2. Kawai T, Sekizuka T, Yahata Y, Kuroda M, Kumeda Y, Iijima Y, Kamata Y, Sugita-Konishi Y and Ohnishi T (2012) Identification of Kudoa septempunctata as the causative

agent of novel food poisoning outbreaks in Japan by consumption of Paralichthys

olivaceus in raw fish. Clin Infect Dis 54: 1046-1052

3. Li Y-C, Sato H, Kamata Y, Ohnishi T and Sugita-Konishi Y (2012) Three novel myxobolid species of genera Henneguya and Myxobolus (Myxosporea: Bivalvulida)

from marine fish in Japan. Parasitol Res 111: 819-826

4. Li Y-C, Sato H, Tanaka S, Ohnishi T, Kamata Y, Sugita-Konishi Y (2013) Characterization of the ribosomal RNA gene of Kudoa neothunni (Myxosporea:

Multivalvulida) in tunas (Thunnus spp.) and Kudoa scomberi n. sp. in a chub mackerel

(Scomber japonicus). Parasitol Res 112: 1991-2003

5. Matsukane Y, Sato H, Tanaka S, Kamata Y and Sugita-Konishi Y (2011): Kudoa iwatai and two novel Kudoa spp., K. trachuri n. sp. and K. thunni n. sp. (Myxosporea:

15

Multivalvulida), from daily consumed marine fish in western Japan. Parasitol Res 108:

913-926

6. Matsukane Y, Sato H, Tanaka S, Kamata Y and Sugita-Konishi Y (2010) Kudoa septempunctata n. sp. (Myxosporea: Multivalvulida) from an aquacultured olive flounder

(Paralichthys olivaceus) imported from Korea. Parasitol Res107: 865-872

16

THE POTENCY OF PHYLLOPLANE SAPROPHYTIC FUNGI ON SHALLOT AS

ANTAGONISTS AGAINST PURPLE BLOTCH DISEASE (Alternaria porri) in

EAST JAVA, INDONESIA

Herry Nirwanto and Tri Mujoko

Faculty of Agriculture UPN “Veteran” East Java, Surabaya, Indonesia

email : heriner@gmail.com

ABSTRACT The purple blotch disease caused by Alternaria porri ( Ell.) Cif.is known as one of a mayor disease

at shallot growing area and is responsible for a great loss.

The obyective of the research is to explore various type of phylloplane and phyllosphere fungi on shallot

crops which have potency as microbial antagonist to A. porri that cause purple blotch disease, and also to

analyse its community.

The Research was conducted at Plant Pest and Disease laboratory, UPN " Veteran" East Java and in rainy

season. The methods used in this research is to conduct survey on shallot crops which applied pesticide.

Sample were taken by purposive sampling to healthy shallot plant among diseased plants. The areas of

survey lied on District of Probolinggo, Malang, Nganjuk and of Kediri at height between 150 - 600 m above

sea level. Antagonism experiment was done by breading pathogen isolate and antagonist fungi isolate in

Potato Dextrose Agar media.

.Results of the research showed that diversity index of saprophytic fungy on shallot crops of Malang isolate

equal to 2,99 and of Probolinggo is, 3,54. The research also found that the isolate of saprophytic fungi of

shallot crop which have potency as antagonist is Trichoderma sp. and Penicillium sp

Key words:, phyllosphere and phylloplane fungi, antagonism, Alternaria porri,

saprophytic, shallot

17

MAXIMIZING THE ESSENTIAL ROLES OF RUMEN MICROBES IN

SUPPLYING NUTRIENTS FOR RUMINANTS

Marjuki Faculty of Animal Husbandry, University of Brawijaya, Malang, Indonesia

Email : marjuki@ub.ac.id

Fibers or forages are the most abundant biomass available in the world. By ruminant

animals, these low quality biomass are potentially to be converted into high quality foods

for human being. Ruminants are very well known to have capability in digesting and

utilizing the low quality biomass for their live and production and convert the biomass into

power, meat, milk, and other products. Indeed, ruminants by themselves are just like other

animals, even they can not digest and utilize fibers or forages that are as their main own

feed, without any essential help of microbes present in their rumen. There is no any

digestive enzymes that can digest fibers or forages, but rumen microbes digest and utilize

the low quality biomass for their live and growth producing high quality microbial cells and

fermentation products volatile fatty acids (VFA) including acetic, propionic, and butyric

acids. These fatty acids are absorbed from the rumen and utilized by ruminant’s body as the

main energy source. In addition, rumen microbial cells flowing to the small intestine are

ready for enzymatic digestion and release their nutrients, especially protein, vitamin and

minerals, which are then ready for absorption to supply the nutrients requirement of

ruminant animals. Thus, rumen microbes play an essential role in helping ruminant to

convert low quality feeds into high quality nutrients to supply most of nutrients required by

ruminants. There is a saying in ruminant feeding that “Feed the rumen microbes first, then

let the microbes feed the ruminants for their live and production”. The first priority in

ruminants feeding is to maximize the rumen microbe’s activity and growth from which

ruminants get their most nutrients supply for their live and production. Rumen microbes

require appropriate supply of substrates and rumen conditions for their maximum activity

and growth. Hence, both requirement of rumen microbial for maximum activity and growth

are directly affected by feeds consumed by ruminants. The feeds do not only function as

nutrients source for rumen microbes, but also creating appropriate rumen conditions for

rumen microbial activity and growth. Thus, feed and its feeding strategy and manipulation

are important to ensure maximum rumen microbial activity and growth, which supply

nutrients for maximum ruminant’s productivity.

Key words: forages, energy, microbes, nutrients, protein, rumen

mailto:marjuki@ub.ac.id

18

Development of effective bioremediation technology

utilizing beneficial biofilms

Masaaki Morikawa

Section of Environmental Biology, Faculty of Environmental Earth Science, Hokkaido University, Japan

In natural environments, bacteria often exist in close association with surfaces and

interfaces. There they form "biofilms", multicellular community structures held together by

extracellular matrices. The biofilms confer on the constituent cells high resistance to

environmental stresses and diverse microenvironments that help generate cellular

heterogeneity. Biofilm-associated cells exhibit specific gene expression, many times

controlled by quorum sensing systems, or dormancy, to allow their increases in resistance.

Thus, forming biofilms is considered a natural strategy of microorganisms to construct and

maintain a favorable niche in stressful environments. Application of biofilms to bio-

production and bio-augmentation process is challenging but it could be a simple and

rational choice (Morikawa, 2006).

Biofilm-associated cells of Pseudomonas stutzeri T102, as compared with that of

planktonic cells, degraded aromatic contaminants naphthalene and survive for longer time

in petroleum-contaminated soils (Shimada et al., 2012). When the fitness of T102 biofilm-

associated cells was tested in natural petroleum-contaminated soils, they were capable of

surviving for 10 weeks; by then T102 planktonic cells were mostly extinct. Naphthalene

degradation activity in the soils that had been inoculated with T102 biofilms was indeed

higher than that observed in soils inoculated with T102 planktonic cells. These results

suggest that inoculation of contaminated soils with P. stutzeri T102 biofilms should enable

bio-augmentation to be a more durable and effective bioremediation technology than

inoculation with planktonic cells.

Biofilms are formed not only on abiotic but on biotic surfaces including plant roots. A

phenol degrading Acinetobacter sp. P23 was isolated from the rhizosphere of duckweed

(Yamaga et al., 2010). P23 rapidly colonized on the surface of sterilized duckweed roots

and formed biofilms, indicating that the conditions provided by the root system of

duckweed are favorable to P23. A long-term performance test using duckweed-P23 system

showed that continuous removal of phenol can be attributed to the beneficial symbiotic

interaction between duckweed and P23. The results in this study suggest the potential

usefulness of colonizing a particular bacterium in the rhizosphere of duckweeds to achieve

efficient and sustainable bioremediation of polluted water.

7. Morikawa M. (2006) Beneficial biofilm formation by industrial bacteria, Bacillus subtilis and related species. J. Biosci. Bioeng. 101: 1-8

8. Shimada K, Itoh Y, Washio K, Morikawa M. (2012) Efficacy of forming biofilms by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology

and its molecular mechanisms. Chemosphere 87: 226-233

9. Yamaga F, Washio K, Morikawa M. (2010) Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated from the rhizosphere of duckweed Lemna

19

aoukikusa. Environ. Sci. Technol. 44: 6470-6474

Microbial diversities in the chemical and organic agricultural soils

Motoki Kubo

1, Andi Kurniawan

1, 2, Kiwako Araki

1, Masaki Mukai

1, Dinesh Adhikari

1, Ir. Sukoso,

Sasmito Djati2, Aida Sartimbul

2

1Department of Biotechnology, Faculty of Life Sciences, Ritsumeikan University, Japan,

2Fishery and Marine

Science Faculty, Brawijaya University

Environmental microorganisms play important roles for material circulation in

agricultural soil. When total carbon (TC) and total nitrogen (TN) in the agricultural soils

were controlled at TC≧30,000 and TN≧3,000, and C/N:8-18, the nitrogen circulation activity was enhanced. Subsequently, inorganic materials such as NH4

+ and NO3

- were also

gradually increased in the soil. Based on these results, soil fertile index (SOFIX) was

constructed (suitable nitrogen and phosphate circulations etc.) for improvement of the

organic agricultural soil condition.

In order to improve the agricultural soil conditions, the bacterial diversities in the

chemical and organic agricultural soils were analyzed by environmental DNA and PCR-

DGGE. The microbial number in the organic agricultural soil was clearly higher than that

in the chemical soil, and the bacterial number was enhanced when TC, TN, and C/N ratio

were controlled (TC≧30,000 and TN≧3,000, and C/N:8-18).

Bacterial diversities in the chemical and organic agricultural soils were different on

the PCR-DGGE gel, and the bacterial species were increased in the improved organic

agricultural field by SOFIX. These results indicate that the bacterial diversity in the

agricultural soil was influence by the contents of chemical and organic materials.

In this meeting, we will show you a creation of suitable organic agricultural soil

condition by controlling microbial diversity with biomass resources. The microbial

diversity in the continuous cropping soil will also be introduced.

1. Matsumiya, Y. Horii S., Matsuno T., and Kubo M., (2013) Soybean as a nitrogen

supplier. In Teck, Edited by James E. Board, 49-60

2. Matsuno T., Horii S., Sato T., Matsumiya Y., and Kubo M., (2013) Analysis of

nitrification in agricultural soil and improvement of nitrogen circulation with autotrophic

ammonia-oxidizing bacteria, Applied Biochemistry and Biotechnology, 169:795-809

3. Horii S., Matsuno T., Tagomori J., Mukai M., Adhikari D., and Kubo M., (2013)

Isolation and identification of phytate degrading bacteria and their contribution to phytate

mineralization in soil, The Journal of General and Applied Microbiology, 59:353-360

20

Exploration of marine bacteria as an alternative source of enzyme

production for industry and biodegradation

Jittima Charoenpanich 1*, Sasithorn Uttatree

2, 3, Khwanlada Kobtrakool

1, Apassara Ketsuk

1,

Wanaree Kaenngam 1, Prachawee Thakolprajak

1, Pairat Ittrat

2, and Jutamas Pantab

4

1Department of Biochemistry, Faculty of Science,

2Environmental Science Program and Centre of Excellence

on Environmental Health and Toxicology (CHE), Faculty of Science, 3Centre of Excellence for Innovation in

Chemistry (PERCH-CIC), Faculty of Science, 4 Bioengineering Program, Faculty of Engineering, Burapha

University, Chonburi 20131, Thailand.

* Corresponding Author: jittima@buu.ac.th

Marine environment is a source of unique microorganisms with great potential for

biotechnological exploitation. Very few studies concerning the isolation and

characterization of marine bacteria have been carried out, and investigations in this field

may lead to many new discoveries. Our group attempts to find the marine bacteria produce

unique characteristic hydrolase for application in industry and biodegradation.

Among 12 marine bacteria isolated from sea sediments of Koh Chan, Samaesan (9

and 24 meters depth), three were found secreting high protease activity. The strains were

identified as Bacillus megaterium, B. subtilis, and Staphylococcus warneri. All enzymes

were stable at alkali pH and active at a broad temperature range 10-80 °C. Metal ions did

not affect the activities of B. megaterium and S. warneri proteases in contrast improve their

activities. Three enzymes were stable in surfactants and hydrophobic solvents. The high

temperature stability, alkaliphilic and ability to work in metal ions, solvents and surfactants

support the potential of these proteases as vigorous biocatalysts for industrial applications.

Microorganisms living in the sea have specially adapted features that allow them to

live and grow in the extreme environment. Many organic compounds have been utilized by

these microorganisms however no report could be found for toxicant removal. We discover

a new benzonitrile-degrader, Staphylococcus sciuri from sea sediments after an oil spill

disaster in Ao Phrao beach, Samet Island. The strain could completely remove benzonitrile

at alkali pH and mesophilic temperature. Moreover, two novel acrylamide-degrading

bacteria (Serratia liquefacian and B. cereus) were also isolated from the sea sediments of

Koh Chan, Samaesan (9 meters depth). Both strains grew well in the presence of

acrylamide as 0.5% (w/v) which is higher concentration than published documents and

prefer acidic pH. Our findings render marine bacteria attractive as an alternative source of

enzyme production for industry and biodegradation.

21

Effects of inoculum size and reactor type on biohydrogen production

from Palm Oil Mill effluent under thermophilic condition

Poonsuk Prasertsan

1, Sompong O-Thong

2 and Jiravut Seengenyoung

1

1 Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla,

Thailand 2 Department of Biology, Faculty of Science, Thaksin University, Phatthalung, Thailand

Biohydrogen production from palm oil mill effluent (POME) in anaerobic

sequencing batch reactor (ASBR) and continuous stirred tank reactor (CSTR) at

thermophilic condition was investigated. The inoculum size of Thermoanaerobacterium

thermosaccharolyticum PSU-2 was tested in the range of 0-30%. The maximum hydrogen

yield of 296 mL H2/g-COD was achieved at 30% inoculums in both ASBR and CSTR, with

the COD removal efficiency of 23%. The COD removal could be increased at continuous

production of hydrogen at the hydraulic retention time (HRT) of 4 days, corresponding to

the organic loading rate (OLR) of 11.3 g COD/ L·day. The COD removal efficiencies of

ASBR and CSTR were 37.7% and 44.8%, respectively.

22

In Vitro thermal adaptation useful for the development of high-

temperature fermentation Kazunobu Matsushita

Director, Research Center for Thermotolerant Microbial Resources, Faculty of Agriculture, Yamaguchi

University, Yamaguchi, Japan

Stable and successful industrial fermentations, in which mesophilic microorganisms

used to be used, could be achieved by consuming a large amount of energy, especially for

cooling and/or sterilization, and also by requiring expensive facilities and/or laborious

labor. Recent global climate change makes more difficult to keep such a stable

fermentation, and thus it is requested to develop a high-temperature fermentation system

with thermally adapted and robust microbes, by which the energy consumption could be

reduced and more efficient productivity provided.

We have isolated a large numbers of thermotolerant useful fermentative microbes such as

acetic acid bacteria and glutamate-producing Corynebacterium glutamicum by collaborating

with Thai groups. In addition, we have also tried to acquire strains adapted to higher

temperature conditions in a laboratory. Thus, we have successfully obtained several thermo-

adapted strains from mesophilic or thermotolerant acetic acid bacteria and also from C.

glutamicum. Of these thermo-adapted strains, TH-3 obtained from Acetobacter pasteurianus

SKU11081)

, ITO-3 from Gluconacetobacter xylinus IFO32882)

, CHM43AD from Gluconobacter

frateurii CHM433)

, and FT-1 from C. glutamicum N244)

have been shown to have a high

potential to make high-temperature fermentation or non-temperature control-fermentation

possible. The former two strains are used for vinegar (acetic acid) fermentation, and the latter two

strains for sorbose and glutamic acid fermentations, respectively.

Aiming at development of the high-temperature fermentation systems, now we have tried to

understand molecular mechanism of their thermotolerance by understanding their genome

modification during the adaptation process1)

, and also to compare their fermentation ability at

different temperature conditions.

In this seminar, I would like to summarize and show their fermentation abilities at high-

temperature condition and also in fementor condition, and to discuss on their ability for the

application to the high temperature fermentations.

1) Matsutani M, Nishikura M, Saichana N, Hatano T, Masud-Tippayasak U, Theeragool G,

Yakushi T, Matsushita K. Adaptive mutation of Acetobacter pasteurianus SKU1108

enhances acetic acid fermentation ability at high temperature. J Biotechnol. 165 (2) 109-

119 (2013)

2) Ito K, Matsutani M, Yakushi T, Matsushita K. Adaptive mutation of Gluconacetobacter

xylinus NBRC 3288 enhances acetic acid fermentation ability (Japanese). Annual meeting

for Nougei-Kagakkai 2014, Kawasaki, Japan; March 28 (2014)

3) Hattori H, Yakushi T, Matsutani M, Moonmangmee D, Toyama H, Adachi O,

Matsushita K. High-temperature sorbose fermentation with thermotolerant Gluconobacter

frateurii CHM43 and its mutant strain adapted to higher temperature. Appl Microbiol

Biotechnol. 95(6) 1531-1540 (2012)

23

4) Nantapong N, Trakulnaleamsai S, Matsutani M, Kataoka N, Yakushi T, Matsushita K.

Characterization of thermotolerant Corynebacterium glutamicum and their genome

analysis.

1st Joint Seminar for New Core to Core Program A. Advanced Research Networks. Centara

Grand & Bangkok Convention Centre, Central World, Bangkok, Thailand, August 10-11

(2014)